Methods for detecting the effect of cell affecting agents on living cells

ABSTRACT

Methods are disclosed for detecting the effects of cell affecting agents on living cells. The method steps include providing living cells that are retained in a micro flow chamber. The micro flow chamber is adapted for either continuous or intermittent flow of solutions or suspensions in intimate contact with the cells. The solutions or suspensions, which contain a cell affecting agent, are then flowed in intimate contact with the cells and at least one effect of the cell affecting agent on the cells is measured by an appropriate detecting means, which is operatively associated with the micro flow chamber.

This application is a continuation of application Ser. No. 07/260,521,filed Oct. 21, 1988 abandoned.

BACKGROUND OF THE INVENTION

1. Field Of The Invention

The present invention relates to methods for detecting the effects ofcell affecting agents on living cells. Solutions or suspensions of cellaffecting agents are flowed over cells and the effects of these agentsare measured.

2. Description Of The Background Of The Invention

Studies of the effect of various cell affecting agents on living cellshave been reported in the literature. See, e.g. Meisner, H. and Tenny,K. (1977) "pH as an indicator of free fatty acid release fromadipocytes," J. Lipid Research, 18:774-776; Nilsson, N. and Belfrage, P.(1979) "Continuous monitoring of free fatty acid release from adipocytesby pH-stat titration," J. Lipid Research 20: 557-560; Reuss, L. ,Weinman, S. and Constantin, J. (1984) "H⁺ and HCO₃ ⁻ transport at theapical membrane of the gallbladder epithelium," pp. 85-96 of Forte, J.,Warnock, D. and Rector, F. Jr. (eds.) Hydrogen Ion Transport inEpithelia, Wiley-Interscience; Zeuthen, T. and Machen, T. (1984) "HCO₃ ⁻/CO₂ stimulates NA⁺ /H⁺ and Cl⁻ /HCO₃ ⁻ exchange in Necturusgallbladder," pp. 97, 108 (ibid.); Handler, J. S. , Preston, A. S. andSteele , R. E. (1984) "Factors affecting the differentiation ofepithelial transport and responsiveness to hormones," FederationProceeding 43: 2221-2224; and Simmons, N. L., Brown, C. D. A. and Rugg,E. L. (1984) "The action of epinephrine on Madin-Darby canine kidneycells," Federation Proceedings, 43:2225-2229. These references disclosethe detection of changes in pH and other electrical potentials by theaddition of cell affecting agents to cells disposed in a relativelylarge amount of medium, i.e., a bulk medium. A disadvantage of thesetechniques is that the pH and other electrical potential measurementsare taken from the bulk medium and do not necessarily reflect the actualvalues immediately adjacent to the cellular membranes of the livingcells. Also, the high ratio of bulk volume to cell volume inevitablydilutes the effects of the cells on the properties of the extracellularmedium. Accordingly, sensitivity is lost or greatly reduced.

Photoresponsive sensors for measuring biochemical systems are disclosedin various patent documents owned by the assignee of the presentinvention. See, e.g., U.S. Pat. Nos. 4,591,550 (Hafeman et al.) and4,704,353 (Humphries et al.); and European Patent Application No.213,825 (Hafeman et al.). U.S. Pat. No. 4,519,890 discloses a flow pHchamber. These patent publications disclose the use of microorganisms tomeasure changes in the environment of the solution to be measured. Thereis no disclosure in these publications of cells in micro flow chambersused to measure of the effects of cell affecting agents. See, also, U.S.Pat. Nos. 4, 737,464 (McConnell et al. ) and 4, 741,619 (Humphries etal. which are likewise owned by the assignee of the present invention.

Various ways of using fluorescence to measure extracellular effects ofliving cells and analytes are disclosed in the literature. e.g., Briggset al. (1985) "Fiber Optic Probe Cytometer" J. Immunological Methods,81:73-81; Hafeman et al. (1984) "Superoxide Enhances Photo BleachingDuring Cellular Immune Attack Against Fluorescent Lipid MonolayerMembranes" Biochemica Biophysica Acta 772:20-28; and U.S. Pat. Nos.4,560,881 and 4,564,598.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide methods fordetecting the effects of cell affecting agents on living cells withgreater accuracy and precision.

It is an additional object of the present invention to provide methodsfor detecting the effects of cell affecting agents on living cells thatavoid the use of large volume, bulk media.

It is a specific object of the present invention to provide methods fordetecting an effect of a cell affecting agent on living cells by: (a)providing living cells that are retained in a micro flow chamber adaptedfor continuous or intermittent flow of a solution or suspensioncontaining the cell affecting agent in intimate contact with the cellsso that the amount of cell affecting agent in contact with the cells maybe controlled; (b) flowing a solution or suspension containing the cellaffecting agent in intimate contact with the cells, thereby producing acell mediated extracellular effect or change in pH, redox potential,cell surface potential or trans-cellular potential; and (c) measuringthe effect of the cell affecting agent by a means for detecting pH,redox potential, cell surface potential or trans-cellular potential thatis operably associated with the micro flow chamber.

In a preferred embodiment of the invention, the cells may be retained inthe micro flow chamber by spontaneous or natural adhesion of the cellsto the internal surface of the flow chamber. Alternatively, the cellsmay be retained in the micro flow chamber by means of a binding agentthat is biologically compatible with the cells. A preferred example ofsuch a binding agent is agarose.

In another preferred embodiment of the present invention, the livingcells may be retained in the micro flow chamber by providing the microflow chamber with a surface having a plurality of wells or depressionsthat act to physically trap the cells on the surface of the microchamber, preferably by gravitational sedimentation. These wells shouldbe of a sufficient width and depth such that the cells remain in themicro flow chamber during ordinary flow rates. The cells may then beremoved from the trapping wells by any appropriate means, including theuse of high flow rates through the micro flow chamber that wash thecells out of the wells or by inversion of the chamber to dislodge thecells from the wells into the flow stream. In an alternative embodiment,the cells may be retained in the flow chamber by trapping them within acompartment of the flow chamber separated by a porous membrane.

The type of micro flow chamber used in the present invention preferablyincludes a chamber of relatively small volume. Preferably this chamberhas a height on the order of 100 μm, though lesser or greater heightsmay be used. The type of micro flow chamber that is preferably used inthe inventive method is of the type disclosed in U.S. Pat. No.4,591,550, the disclosure of which is incorporated herein by reference.An argon laser may be used as a source of energy in place of the lightemitting diodes disclosed in U.S. Pat. No. 4,591,550. Alternatively, themicro flow chamber useful in the present invention may be of the typedisclosed in pending U.S. patent application Ser. No. 876,925, filedJun. 20, 1986, which is owned by the assignee of the present invention,the disclosure of which is incorporated herein by reference. Mostpreferably the micro flow chamber to be used in the present inventionincludes a silicon semiconductor electrode on or near which the livingcells are retained. By means of this electrode, the various electricaleffects caused by the cell affecting agent may be detected or measured.The micro flow chamber to be used in the present invention shouldpreferably provide for both intermittent and continuous flow ofsolutions or suspensions, since either intermittent or continuous flowof solutions or suspensions may be used in practicing the presentinvention.

The present invention may be used in conjunction with either eukaryoticor prokaryotic cells, so long as the particular cells are capable ofbeing retained in the micro flow chamber. In addition, a wide variety ofcell affecting agents may be used, including irritants, drugs, toxins,other cells, receptor ligands, immunological agents, viruses, pathogens,pyrogens, and hormones.

A wide variety of effects caused by the cell affecting agents may bedetected or measured according to the present invention. Preferredeffects include the pH, redox potential, and other electrical propertiesof the solution or suspension that flows in intimate contact with theliving cells in the micro flow chamber, such as cell surface potentialand transcellular potential. These effects may be measured or detectedby a variety of conventional means. For example, pH can be detected bymeasuring the fluorescence or absorbance of a pH sensitive dye such asfluorescein or phenol red in the extracellular medium or fixed to a partof the chamber. In a similar manner other dyes can be used to detectredox potential.

The present invention also includes methods of identifying microbes,methods of screening for the activity of drugs, methods for detectingtoxic substances and methods for detecting intercellular reactions. Inthese various methods, solutions or suspensions containing the desiredcell affecting agent are flowed in intimate contact with the livingcells retained in the micro flow chamber. The effect(s) of the cellaffecting agent on the cells are then measured and provide the means bywhich bacteria may be identified, drugs screened, and toxins andintercellular reactions detected.

Certain preferred embodiments of the present invention are discussedbelow in more detail in connection with the drawings and the detaileddescription of the preferred embodiments. These preferred embodiments donot limit the scope or nature of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-3 represent schematic cross-sectional views of micro flowchambers useful in practicing the preferred inventive methods.

FIG. 4 represents a schematic diagram of apparatus useful in practicingthe preferred inventive methods.

FIG. 5 represents a schematic diagram of a degasser useful in practicingthe preferred inventive methods.

FIGS. 6-16 represent the experimental results obtained in connectionwith Examples 1, 2, 4, 6, 9, 11 and 17.

FIG. 17 represents a schematic plan view of wells for retaining cellsuseful in practicing the preferred inventive methods.

FIG. 18 represents results of exposing gram negative bacteria to anantibiotic which has an effect and one that has no effect on the gramnegative bacteria.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to FIG. 4, the instrumental setup of the preferred embodimentpreferably consists of one or more syringe drives or pumps, a degassingchamber, a selection valve or injection loop valve, a flow chamber, areference electrode reservoir, and the associated electronics requiredfor the silicon semiconducting electrode sensor and data processing.

The syringe drives provide the solutions or suspensions (i.e., themedium) to the flow chambers at controlled flow rates. Both the flowrate and the on/off cycle of the syringe drives can be controlled bymeans of a computer during the data acquisition phase.

Referring to FIG. 5, the degassing chamber consists of the length ofsilicone rubber tubing through which the solutions or suspensions pass.The outside of the tubing is maintained in a reduced pressure airatmosphere (approximately 2/3 atmospheric pressure). A degassing chamberis preferred because the solutions or suspensions in the syringes andtubing are at room temperature and the flow chamber is commonly at 370°C. As the room temperature medium reaches the flow chamber it warms to370° C. resulting in the formation of bubbles in the chamber. Bubbles inthe flow chamber interfere with measurements in several ways. They cancause a high resistance path to the reference electrode resulting in anincrease in noise in the photocurrent signal. They can also alter theaqueous volume in the chamber thus changing the buffer capacity of thechamber. For a given cellular metabolic rate, the rate of change of pHin the chamber will vary with the number and size of the bubbles.Bubbles form in the chamber and grow with time. When they reach acertain size they dislodge and are carried away in the flowing stream.This can create low frequency noise in the buffer capacity of the flowchamber.

The selection valve allows the user to direct the flow from one of twosyringes into the flow chamber. An alternative is an injection loopvalve. This allows the user to inject a bolus of solution or suspensioninto the flow stream without interrupting the flow. Typically when theinjection loop valve is used, one syringe is loaded with medium and usedto perfuse the stream by means of the injection loop. The volume of theagent solution or suspension and the time of exposure of the cells tothe agent can be varied by varying the injection loop size and the flowrate of the perfusion stream.

FIGS. 1-3 illustrate a preferred micro flow chamber 1 having a siliconsensor 2 with an inlet port 3 and an outlet port 4. In FIG. 1, cells 5are adhered in a monolayer to the upper surface of silicon sensor andthe response on the silicon sensor is modulated by laser light 6.Alternatively, the cell can be retained adjacent the silicon sensor by apermeable membrane. In operation, a solution containing a cell affectingagent enters through inlet port 3 and flows over cells 5 in the microflow chamber 1. The solution exits outlet port 4 where the exitingsolution is in electrical contact with a reference electrode. The localresponse on the upper surface of the silicon sensor 2 is modulated bylaser light 6 and measured. FIG. 2 illustrates wells 7 that trap orretain therein cells during slow flow rates and from which the cells maybe flushed out of the flow chamber during fast flow rates.

The flow chamber is preferably a thin channel bounded on the bottom bythe silicon sensor 2, and on the top by an indium-tin-oxide (ITO) coatedglass cover slip 8. Adherent cells may also be grown on the surface ofthe cover slip instead of the silicon semiconductor electrode. Thespacing between the silicon sensor and the cover slip is approximately100 μm. A platinum wire (not shown) penetrates the plastic housing ofthe flow chamber to make electrical contact with the ITO controllingelectrode. Electrical contact with the silicon sensor is made via themetal baseplate 9 of the flow chamber. Teflon inlet and outlet tubes, 3and 4, penetrate the plastic housing and allow medium to flow throughthe flow chamber. The outlet tube 4 terminates in a reservoir thatcontains a Ag/AgCl reference electrode. Thus the outlet tube 4 acts as asalt bridge to allow measurement of the potential of solutions orsuspensions in the flow chamber. The entire flow chamber is mounted on ahollow metal support that is maintained at constant temperature,typically 370° C., by a temperature controlled circulating water bath10. The cover slip 8 is removable and may be maintained in operativeposition by means of removable silicon rubber gaskets 11 and retainingmembers 12.

Two different instruments may be preferably used for the purpose ofilluminating the silicon sensor to generate a suitable photoresponse: alaser instrument and a light emitting diode (LED) instrument.

In the laser instrument, the flow chamber is mounted on a lightmicroscope stage. The beam from a 150 mW argon ion laser is directedthrough a 10 kHz mechanical chopper and into a beam expander thatgenerates a beam approximately 2.5 cm in diameter. The beam then passesthrough a polarizing filter (variable attenuator) and is directed bymirrors through a quadruple knife edge adjustable aperture into thebarrel of the microscope. In this configuration, one can see the cellsin the flow chamber through the microscope and can direct a square orrectangular probe beam of any desired size to any region of the siliconsurface in the flow chamber. This is particularly useful whennonconfluent cell cultures are used. It allows one to direct the probebeam to regions of greatest cell density. In addition one can controlfor instrumental drift by directing the beam to regions where no cellsare visible.

In the LED instrument, two flow chambers are mounted side by side on atemperature controlled plate. For illumination, four fiber optic lightguides for each chamber penetrate the temperature controlled plate toilluminate the silicon sensor from underneath (the side opposite thesurface in contact with the medium). The four fibers are aligned so asto have one near the inlet tube, one near the outlet tube, and twoequally spaced between the inlet and the outlet. This instrument ispreferably used with cells that grow to a reasonably consistent anduniform density. However, it still gives some latitude in pickingregions of greatest cell density. One end of the optical fibers butts upagainst the silicon sensor and the other end is coupled to an infraredLED. Each fiber is 1.5 mm in diameter. Data can be collected from eightsites (four for each of the two chambers) simultaneously every fourseconds.

Living cells may be adhered to the chamber in a variety of ways. Fornaturally adherent cells, the cells may be grown either on the surfaceof the silicon sensor or the cover slip in an incubator. The flowchamber is assembled while keeping the cells moist. Flow is establishedquickly to keep the cells from over acidifying the medium or consumingall of the oxygen in the chamber. For non-adherent cells, the cells maybe mixed with an agarose solution at 370° C. and plated onto the surfaceof the silicon sensor or cover slip in an approximately 50 μm thicklayer in a humid atmosphere. The silicon sensor is then refrigerated forabout 15 min. to solidify the agarose. The flow chamber is thenassembled and used.

Collagen and gelatin may also be used in conjunction with or in place ofagarose. Fibronectin, chondronectin, laminin or other similar substancesmay optionally be used in adhering the living cells to the surface ofthe micro flow chamber. Alternatively, the living cells may be dispersedon or in biologically compatible microcarrier beads.

The generally preferred inventive method may be practiced as follows.The chamber is assembled with living cells retained therein and attachedto the fluidics in the instrument. Flow (typically 100 μL/min) of a lowbuffer capacity solution or suspension is established and the signalfrom the sensor is allowed to stabilize. Some drift is seen initiallydue to warming of the chamber and equilibration of the chamber with thenew medium. Once stable, flow in the chamber is halted and the siliconsensor signal is monitored. A decrease in potential reflects thedecrease of pH in the chamber due to cellular metabolism. The rate of pHdecrease is a measure of the metabolic rate of the cells. The pH isallowed to drop far enough to obtain enough data points to accuratelydetermine the slope of the line (within a few percent error). Typicallythis reflects a pH drop of approximately 0.1 to 0.5 pH units andrequires approximately 1 to 4 minutes. Flow is then restarted and the pHreturns to the initial value. This sequence is repeated untilreproducible slopes are obtained.

In another preferred embodiment, medium is flowed continuously over thecells and the potential is measured at a plurality of sites. If one siteis located upstream of the direction of flow from another and there areintervening cells, there will be a pH difference between the two siteswith the upstream site being less acidic. This pH difference is due tothe metabolic action of the cells on the medium as it traverses thespace between the sites. The magnitude of this pH difference, asdetected by the silicon semiconductor electrode, is a measure of themetabolic rate of the cells located between the two sites.

For testing cell affecting agents, two strategies may be advantageouslyemployed. In one approach, two syringe drives are used. First metabolicrates are determined as described above with a control medium. Then thesyringe drive with the control medium is turned off and a syringe drivewith the same medium plus the cell affecting agent is turned on. Anappropriate selection valve directs the flow from either syringe driveto the flow chamber. Metabolic rate measurements are then taken in thepresence of the cell affecting agent. The flow can be switched back tothe control medium to observe recovery of the cells from the effect ofthe cell affecting agent.

In a second approach, an injection loop valve is used. Metabolic ratesare determined as described above. Once they are stable, the cellaffecting agent is introduced into the injection loop, and the valve isturned such that the cell affecting agent is introduced into the flowingmedium stream. Metabolic rates can then be measured in the presence ofthe cell affecting agent and can be followed after the agent is washedout of the chamber. This technique is particularly suited for testingfor cell responses to brief exposure to cell affecting agents.

If the living cells to be used are not naturally or spontaneouslyadherent to a surface of the micro flow chamber, means of retaining thecells in the chamber must be used. Three desirable ways of achievingthis result are the provision of wells capable of physically retainingthe cells, the provision of a compartment in the micro flow chamber thatretains the cells by means of a membrane that is porous or permeable tothe cell affecting agent in the solution or suspension, and the use ofbiologically compatible adhering agents.

In the embodiment employing wells, the floor of the flow chambercontains the wells. The wells may be circular in cross-section or may betrenches aligned perpendicularly to the direction of flow. The smallesthorizontal dimension of the wells should be at least one cell diameter,preferably no more than several cell diameters. The depth of the wellscan be several cell diameters. Wells 50 μm in diameter and 50 μm deepare acceptable. The use of wells to retain the cells is well adapted forthe analysis of multiple batches of cells by employing the followingcycle of operations.

The first step is loading cells into the wells. At sufficiently slowflow rates cells will sediment into the wells and remain trappedtherein.

Next, the cellular behavior is analyzed. The composition of the mediumflowing over the wells can be altered according to the desiredanalytical protocol. Compounds from the medium reach the cells in thewells by a combination of diffusion and convection.

Finally, the cells are flushed out of the wells. At sufficiently highfluid flow rates, vortex behavior in the wells becomes strong enough tocause the cells to be resuspended and to be swept out of the wells andout of the chamber. This clears the chamber for the next loading offresh cells.

There are at least two important advantages to the well method. Firstthe method of immobilization is nonperturbing. Second, the system iseasily automated to provide repetitive analyses of fresh batches ofcells in the same micro flow chamber.

In the embodiment employing biologically compatible adhering agents,non-adherent cells may be retained in the flow chamber in a very thin(preferably 50 μm thick) layer of agarose covering the floor of a flowchamber having a total depth 100 μm and surface area about one cm².Medium flows through the 50 μm gap remaining between the ceiling of thechamber and the top of the agarose layer.

The thinness of the agarose layer permits rapid and virtually unimpededaccess of the immobilized cells to the components of the medium flowingover the agarose. Transport between the cells and the flowing medium isdiffusive. Diffusion times on the order of one to ten seconds are to beexpected for most solutes.

The cell retaining wells are preferably formed on the surface of thesilicon semiconductor electrode or sensor. They may be formed asindentations in the surface of the electrode or by application of a gridstructure on top of the upper surface of the electrode. The gridstructure is preferably formed of an electrically compatiblesemiconducting material, i.e. silicon, but may be formed of electricallyneutral materials. For example, a nylon mesh comprised of 25 μm diameterthreads woven to produce 25 μm square holes may be glued to the surfaceof the silicon semiconductor electrode with a spray adhesive. Theadhesive forms widely dispersed fine droplets on the silicon electrodeand therefore only attaches the nylon mesh to the surface at a fewdiscrete points. In this manner, the nylon mesh may be attached to thesilicon electrode without totally coating the silicon electrode withadhesive and thereby destroying the photoresponse of the electrode.

The present invention may be used with a wide variety of prokaryoticand/or eukaryotic cells. Examples of such cells include humankeratinocytes, murine L fibroblastic cells, canine MDCK epithelialcells, hamster BHK fibroblastic cells, murine CTLL lymphocyte cells andbacteria. In general, any living cells that can be successfully retainedwithin a micro flow chamber may be used.

The present invention may be used with a wide variety of cell affectingagents. Examples of such cell affecting agents include irritants such asbenzalkonium chloride, hydrogen peroxide, 1-butanol, ethanol, and DMSO;drugs such as valinomycin, amiloride and theophylline; hormones such atT₃ and T₄, epinephrine and vasopressin; toxins such as cyanide,endotoxins and bacterial lipopolysaccharides; immunological agents suchas interleukin-2 and various other types of cell affecting agents suchas phorbol myristate acetate, magnesium chloride, other cells, receptorligands, viruses, pathogens and pyrogens. This invention alsoencompasses the measurement of the effects of water immiscible cellaffecting agents such as particulate matter, greases, and oils on cells.These substances can be delivered to the vicinity of cells by aqueous ornon-aqueous fluids.

The present invention includes methods for identifying microbes such asyeast, bacteria, and fungi. In these inventive methods, the bacteria tobe identified are trapped or retained within the flow chamber. Apredetermined set of solutions and/or suspensions are then sequentiallyflowed through the micro flow chamber such that they come in contactwith the bacteria. Each of these solutions and/or suspensions containsan ingredient that produces a particular response from known bacterium.When the solution and/or suspension containing this ingredient arecontacted with bacteria to be identified, the bacteria in the micro flowchamber either produce a characteristic response or produce no response.The response or absence of response is then measured by means of thesilicon semiconducting electrode. The response or lack of response tothe predetermined set of solutions and/or suspensions may then becompared to the response of known bacteria to the predetermined set ofsolutions and/or suspensions in order to positively identify thebacteria being tested.

The present invention also includes methods for screening for theactivity of a drug. In these inventive methods, living cells that areresponsive to the drug to be screened for are trapped or retained withinthe micro flow chamber. A solution or suspension suspected of containingthe drug may then be flowed into intimate contact with these livingcells. Upon contact with the living cells, the solution or suspensionsuspected of containing the drug will produce a response in the livingcells or produce no response at all. This effect or lack of effect ofthe drug on the living cells is then measured by means of the siliconsemiconductor electrode. Such drugs may include antibiotics activeagainst microbial cells. The presence or absence of the expected effectmay be used as a means for screening solutions or suspensions for thepresence of a drug.

The present invention also includes methods for detecting toxicsubstances. In these inventive methods, living cells that are responsiveto the toxic substance being tested are trapped or retained within themicro flow chamber. The solution or suspension suspected of containingthe toxic substance is then flowed into intimate contact with the livingcells. Any reaction or lack of reaction of the living cells to thesolution or suspension is measured, thereby providing a means fordetecting the presence of the suspected toxic substance.

The present invention also includes methods for detecting intercellularreactions. In these inventive methods, two sets of living cells areprovided within the micro flow chamber. A solution or suspensioncontaining an agent that affects the first set of cells is flowedthrough the micro flow chamber. This causes the first set of cells toelaborate a second cell affecting agent. The second cell affecting agentin turn causes a response in the second set of living cells. Thisresponse is measured by means of the silicon semiconductor electrode. Inthis way, the intercellular reactions of different sets of living cellsmay be measured. The first and second sets of cells may be of the sametype or may be of different types.

EXAMPLE I

Keratinocytes are the cells that generate the epidermis. Studies havebeen done to assess the response of these cells to irritants that mightcontact the skin in the workplace, in cosmetics, and elsewhere. Forexample, Shopsis and Eng (Shopsis, C. and Eng, B. (1988) in AlternativeMethods In Toxicology, Vol. 6)) determined the concentration of anirritant necessary to inhibit protein synthesis by 50% in a 48-hourincubation of keratinocytes with the irritant. The ranking of a seriesof detergent irritants by this method agreed well with the rankingsobtained by the standard in vivo Draize test of ocular irritancy(Draize, J. , Woodard, G. and Clavery, H. (1944) J. Pharmacol. Exp,Ther., 82:377-390).

Normal human keratinocytes were obtained from Clonetics, Inc. (SanDiego, Calif.) and cultured according to the vendor's instructions. Thecells were grown to between 50% and 100% confluency on the conductiveindium-tin-oxide surfaces of the glass cover slips used in the cell flowchamber. The irritants tested were benzalkonium chloride (BAC; acationic detergent), hydrogen peroxide (H₂ O₂), 1-butanol (BuOH),ethanol (EtOH), and dimethyl sulfoxide (DMSO).

The cover slip was assembled in the chamber, and flow of medium wasinitiated. Clonetics provided a low-buffered modification of theirKeratinocyte Growth Medium for this purpose; it contained neitherbicarbonate nor HEPES buffer.

The flow of medium was controlled by a computer-interfaced syringe pump.Flow was alternately on for 200 sec and off for 200 sec. During theflow-off time, the metabolic rate was determined as the slope of thetrace of sensor potential (i.e., pH) vs. time.

Irritants were injected via a 300 μl sample injection loop (of the typeused in liquid chromatography) at the beginning of a flow-on period.

The flow rates, on the order of 100 μl/min, were adjusted so that theirritant was present in the cell chamber during the next flow-off periodbut had been cleared by the second flow-off period. Typically, sampleswere injected at 800-sec intervals, allowing two determinations ofmetabolic rates for each injection (one with the irritant present, oneafter it had cleared).

After a stable metabolic rate had been established in the absence ofirritants, irritants were injected as described above, working from lowto high concentration. Often there were two injections of eachconcentration. An entire experiment took three to five hours, though aresponse to each dose of irritant was obtained 400 sec after sampleinjection. As a control, the metabolic rates of one set of keratinocytesthat were administered no irritants were measured over several hours.

The metabolic rate of the control cells remained stable throughout theexperiment, with a coefficient of variation of about 6%. FIG. 6 showsthe results for the five irritants, plotted as metabolic rate vs. thelogarithm of irritant concentration. Metabolic rate is expressed as apercent of the value prior to the introduction of irritant. When twosuccessive doses were administered at the same concentration, both setsof data are presented. The large differences in such points at, e.g., 30μg/ml BAC, represents a real decrease of cellular metabolic rate betweenthe two administrations, not mere scatter in the data. The H₂ O₂ wasmixed with medium several hours before administration, so that actualconcentration of this irritant may have been less than indicated, due toredox reactions with compounds in the medium as well asdisproportionation reactions. For some irritants the metabolic rate roseabove the control level for irritant concentrations just below thosethat were sufficient to inhibit metabolism.

FIG. 7 is a comparison of measurements taken during the presence of theirritant and after washout. The closed symbols represent rates obtainedduring the presence of the irritants on the cells; the open symbolscorrespond to the rates obtained immediately after the irritants werewashed out. For one irritant, BAC, no recovery was obtained; in fact,metabolism continued to decrease with each measurement after the higherdoses. For ETOH, there was substantial recovery.

The irritant strength of these compounds has been given the followingclassification by in vivo tests such as the Draize test (see, e.g.,Dubin, H. and Chodgaonkar, R. (1987) In Vitro Toxicology, 1:233-240):

    ______________________________________                                        Compound           Strength                                                   ______________________________________                                        BAC                Severe                                                     H.sub.2 O.sub.2    Severe                                                     BuOH               Moderate                                                   EtOH               Mild-Moderate                                              DMSO               Mild                                                       ______________________________________                                    

The ranking of the concentrations necessary to depress metabolism inthis example agrees well with that obtained by the in vivo tests.

The inventive method allows flexible control of both the duration andconcentration of exposure of the cells to the irritants. Furthermore, itpermits one to monitor the effects of these agents on cell metabolismboth while the irritants contact the cell and then later, after theirritants have been washed out. Thus, the kinetics of recovery from theinsult can be determined along with the main inhibition analysis.

This example demonstrates the feasibility of performing rapid in vitrotoxicological assays with the inventive method. Responses to acuteexposure have been obtained in a few minutes. Further automation andexploitation of parallelism would bring the time for an entireexperiment down to that amount of time. This is a significant advantagein speed and capability over existing in vitro methods.

EXAMPLE 2

Valinomycin is a peptide antibiotic that exerts its effects by insertingitself into cell membranes and rendering them permeable to potassiumions. It is commonly used in research applications where membranepermeability to ions is important. Within no more than a few minutesafter being exposed to 100 μm valinomycin, the metabolic rate of L cellsrises significantly; at its peak it may be more than double the initialrate. Following the peak, the rate drops.

FIG. 8 shows the response to a brief pulse of valinomycin according tothe inventive method (75 μl injected into the medium stream; flow ratesabout 100 μL/min). In other experiments (graph not shown) the sameconcentration of valinomycin was administered continuously for 8 minutesand the metabolic activity rose to a peak of approximately 2.4 times theinitial value four minutes after the start of exposure. It returnedapproximately to the initial value within 20 minutes of the removal ofthe antibiotic. This biphasic response may be an initial expenditure ofcellular metabolic energy to attempt to maintain electrolytehomeostasis, followed by the toxic effects of the cells' failure toaccomplish this.

EXAMPLE 3

L cells were grown to confluence on the silicon sensor surface of theflow chamber and alternately perfused with normal medium and mediumcontaining 10 nM each of the thyroid hormones triiodothyronine (T₃) andthyroxine (T₄). In one experiment when the hormone was first introduced,the metabolic rate increased by 12% and decreased to 9% below theinitial level when the hormone was withdrawn. This is in accordance withthe expectation that the hormone increases the overall metabolicactivity of the cells.

EXAMPLE 4

Phorbol myristate acetate (PMA) is one of the tumor-promoting phorbolesters. Among its effects are an increase in cellular cyclic AMP levelsand the deacylation of phospholipids to produce arachidonic acid; it isknown to interact with protein kinase C (see, e.g., Daniel, L. (1985)Phosphatidylinositol Turnover in MDCK Cells, in Inositol andPhosphoinositides: Metabolism and Regulation, (eds.) Bleasdale, J.,Eichberg, J., and Hauser, G. Humana Press). MDCK cells (Madin-DarbyCanine Kidney) are an epithelial line that retain many of the functionalcharacteristics of kidney tubule epithelia. One characteristic is that aconfluent monolayer of these cells forms a relatively tight seal, bothelectrically and to the diffusion of ions, separating the aqueousregions on the two sides of the cells.

The sealing property of the MDCK cells implies that when the cells aregrown on the surface of the silicon sensor, the signal does notnecessarily represent merely the pH of the fluid in the flow chamber. Itcontains information on the pH of the small aqueous compartment betweenthe cells and the sensor, and it contains information abouttrans-cellular potentials, which are essentially batteries in serieswith the electronic circuit of the sensor. In cells that do not form atight seal, the sub-cellular pH is essentially identical to the flowchamber fluid pH and any trans-cellular potentials are shorted out asfar as the sensor is concerned.

FIG. 9 shows the strong and rapid response that was observed when mediumcontaining 10 ng/ml PMA was introduced into the system. The steady-statepotential reported by the sensor (with fluid flow on) decreasedgradually from the time the sensor was activated until the PMA wasintroduced. At that time it dropped quickly to a minimum about fortyminutes later when the PHA was removed, then increased and finallyreturned to a value near its initial one by the end of the experiment.This signal is a composite of the trans-cellular potential and thesub-cellular pH (changes in the latter corresponding to 59 mV per pHunit).

The introduction of PMA coincided with a sharp rise in the metabolicrate of the cells, more than a doubling at the peak near an hour later.Similar experiments with MDCK cells grown on the cover-slip surface ofthe chamber give no information about trans-cellular potentials but alsoindicate an increase in metabolic rate upon the addition of PMA (datanot shown).

The metabolic rates determined from cells grown on the sensor surfaceare more sensitive to the treatment of the cells and generally lessstable than those from cells grown on the cover slips. This isunderstandable in terms of the small volume of the sub-cellular aqueouscompartment compared to the volume of the chamber; small changes intransport of carbon dioxide and protons across cell membranes can causelarger changes in pH in smaller volumes.

The effects of PMA on steady-state potential are particularlyinteresting in light of the fact that PMA is known to disrupt the tightjunctions responsible for the resistive sealing between these cells. Onewould expect, therefore, PMA to short out the effects of trans-cellularpotentials and to make the composition of the sub-cellular space morelike that of the general flow chamber fluid.

EXAMPLE 5

Cyanide (CN⁻) is a anion that binds to the oxygen combining site ofcytochrome oxidase and inhibits electron transport (thereby inhibitingcell respiration). Cells become glycolytic after being exposed to CN⁻. LCells (a mouse fibroblast cell line) were grown to confluence on thesilicon sensor surface of the flow chamber and exposed to an injectionof 100 μM sodium cyanide. Within minutes, the metabolic rate increased39% above the rate when the compound was introduced, and then the ratebegan to gradually decrease. At this point, the cells were rounded upand beginning to die.

EXAMPLE 6

BHK cells (a fibroblast line from baby hamster kidneys) were grown onthe silicon sensor surface of the flow chamber and alternately perfusedwith normal medium and medium containing 0.6 or 0.06 E.U. of bacteriallipopolysaccharide endotoxin. As shown in FIG. 10, the introduction of0.06 E.U. of endotoxin (with 0.001% BSA as a protein carrier) resultedin a 31 to 36% increase in metabolic rate over the rate obtained whenthe cells were in the presence of medium only (control value). Themetabolic rate remained unchanged (with respect to the rate observedwhen cells were in the presence of medium only) when medium with 0.001%BSA (bovine serum albumin) or medium with 0.01% BSA plus 0.6 E.U. ofendotoxin was introduced. This result may reflect a biphasic response ofBHK cells to endotoxin. This is frequently seen for receptor mediatedstimulation of cells. A low dose of stimulant gives a response whereas avery high dose can give no response or an opposite response compared toa low dose.

EXAMPLE 7

Amiloride is a diuretic drug that has been used extensively as aninhibitor of the Na⁺ channel of transporting epithelial cells present inthe kidney and the toad bladder. Taub et al. demonstrated that the MDCKcell line possesses an amiloride-sensitive Na⁺ channel characteristic ofthe cells present in the distal tubule of the kidney (J. CellularPhysiology, 106:191-199 (1981)). When MDCK cells were grown on the coverslip portion of the flow chamber, the introduction of 1.5 MM amilorideresulted in a gradual increase of metabolic rate up to 16% above therate observed for medium only. Once exposure to the drug was ceased, therate returned to the original control value and then gradually continuedto decline.

When MDCK cells were grown on the silicon sensor surface of the flowchamber, the introduction of 1.5 mM amiloride resulted in an immediate20% decrease in apparent metabolic rate with respect to the rateobserved in the presence of medium only.

Interpretation of the data collected from MDCK cells was made difficultby the fact that as cell respiration made the medium more acidic (aftermedium flow was stopped), the cells responded to the change in pHthereby resulting in a change in the original metabolic rate, to whichthe cells could also respond. This complicated cell response resulted ina multiphasic curve when medium flow was stopped for a period of time.In the case where the cells were grown on the silicon sensor surface,this phenomenon was particularly dramatic.

EXAMPLE 8

Theophylline (a diuretic, cardiac stimulant, and smooth-muscle relaxant)has been shown to inhibit phosphodiesterase, which results in anincrease in the amount of CAMP in the cell. When MDCK cells were grownon the silicon sensor surface, the introduction of 10 mM theophyllineresulted in a 47% decrease in the metabolic rate with respect to thecontrol value. After the theophylline containing medium was removed, themetabolic rate returned to the control value within ten minutes. Whilethe therapeutic level of theophylline (111 μM) introduced to MDCK cellsgrown on the silicon sensor surface resulted in an approximate 10%decrease in metabolic rate, a 28% increase in rate with respect to thecontrol values was observed when theophylline was removed from the cellenvironment.

MDCK cells grown on a cover slip demonstrated a similar decrease in themetabolic rate (59%) when the cells were exposed to 10 mM theophylline,although a gradual increase in rate was observed (up to only 48% belowthe control value). Once the theophylline was removed from the cellenvironment, the metabolic rate increased 30% over the control value.

EXAMPLE 9

MDCK cells are known to have epinephrine receptors on their basolateralsurface (the surface of the cells which attaches to the substrate onwhich they are growing). Therefore, when MDCK cells are grown on thesilicon sensor surface, the epinephrine receptors are oriented on thesensor side of the cellular monolayer. When medium containing 3 μMepinephrine was flowed over the cell monolayer, the epinephrine had todiffuse across the cell layer to reach the receptor. The primaryintroduction of medium plus epinephrine did not significantly alter themetabolic rate with respect to the control rate. However, as shown inFIG. 11, additional exposure to medium plus epinephrine resulted in a20% increase in metabolic rate. The metabolic rate remained 12% higherthan the control rate after the epinephrine containing medium wasremoved from the cell environment. An additional 7% increase inmetabolic rate was attained when the cells were once again exposed tothe epinephrine containing medium (i.e. a 20% increase in metabolic rateover the control rate).

EXAMPLE 10

Another method to immobilize cells is to encapsulate them in an agarosegel. MDCK cells in a confluent T-75 flask were trypsinized in order toremove them from their growth container. The cells were centrifuged at2000 RPM for 5 minutes and the supernatant was discarded. The cells wereresuspended in 5 mL of medium and centrifuged for 5 minutes at 3000 RPM.The supernatent was again discarded, and the cells were resuspended in500 μL of 1. 2% sea plaque agarose from FMC (in medium) . The cells werecentrifuged for 3 minutes at 3000 RPM to pellet the cells. A 2 μLaliquot was taken directly from the cell pellet and spread over anapproximate area of 40 mm² on the silicon sensor surface (in a partiallyassembled chamber). The chamber was refrigerated for 15 minutes in orderto allow the agarose to gel. The assembly of the flow chamber was thencompleted and the cells were alternately perfused with medium only,medium plus 208 μM epinephrine, or medium plus 3 μM epinephrine. Thepresence of 208 μM epinephrine resulted in a 3% decrease in themetabolic rate with respect to the rate observed for medium only.However, as shown in FIG. 12, the removal of the epinephrine from thecell environment resulted in a 14% increase in metabolic rate over thecontrol rate. Similarly, the introduction of 3 μM epinephrine to thecells resulted in a 4.5% decrease in metabolic rate with respect to therate observed immediately after the higher epinephrine concentration wasremoved. When the 3 μM epinephrine was removed from the cellenvironment, the metabolic rate increased 17% over the rate observedprior to its introduction.

EXAMPLE 11

CTLL cells (a cytotoxic T-Cell line from mice) are a nonadherent cellline that requires the presence of Interleukin-2 (IL-2; an immunemodulator) to remain viable. Two mL of cells were removed from aconfluent T-25 flask of CTLL cells and centrifuged to pellet the cells.The supernatant was discarded and the cells were resuspended in 300 μLof a 2% agarose solution (in medium) . The cells were again centrifugedto a pellet, and a 2 μL aliquot was removed from the cell pellet andspread over an approximate 40 mm² circle on a cover slip that was housedin a humidified chamber. The cover slip chamber was then refrigeratedfor 20 minutes to allow the agarose to gel. The cover slip was removedfrom the humidified chamber and placed in a f low chamber. Mediumcontaining 20 units/mL of IL-2 or medium only was alternately flowedacross the cells. When the medium containing IL-2 was removed, as shownin FIG. 13, the metabolic rate began to decline with respect to thecontrol rate. The rate continued to decline until medium containing IL-2was reintroduced to the system, at which time the rate began to increase(although the rate did not return to the control rate value). At thistime, the cells were beginning to detach from the agarose gel. Asexpected, as the cell density decreased, so did the rate ofacidification of the chamber.

EXAMPLE 12

L cells were grown to confluence on the silicon sensor surface of theflow chamber and alternately perfused with normal medium and mediumcontaining 0.6 E.U. of bacterial lipopolysaccharide endotoxin. In oneexperiment the introduction of endotoxin increased the initial metabolicrate 20% above the rate observed for medium only, and a second exposureof the cells to endotoxin increased the rate 15% above the rate observedfor medium only. In both cases, the rate returned to the initial valuewhen the endotoxin containing medium was removed (within a few minutes).

EXAMPLE 13

Oxytocin is the principal uterus-contracting and lactation-stimulatinghormone of the posterior pituitary gland. The introduction of 1 μMoxytocin to MDCK cells grown on a cover slip did not result in anyimmediate change in the metabolic rate with respect to the controlvalue, although the metabolic rate did gradually decrease (up to 10%).The removal of the oxytocin from the cell environment did notimmediately return the cell metabolic rate to its control value,although within 10 minutes 100% recovery was attained. This was acontrol experiment since MDCK cell do not have oxytocin receptors, andoxytocin is a nonapeptide which differs from vasopressin by only twoamino acids. MDCK cells do have vasopressin receptors. This was notnecessarily the best control experiment because, as shown below, onlycells grown on the silicon sensor surface demonstrated a response tovasopressin.

EXAMPLE 14

The introduction of 0.1 μM vasopressin (antidiuretic hormone) to MDCKcells grown in the silicon sensor surface resulted in a 14% increase inmetabolic rate over the control values. The addition of 1 μM vasopressinimmediately after the 0.1 μM vasopressin resulted in another increase inthe metabolic rate (19% over the rate attained in the presence of 0.1 μMvasopressin, which is a 42% increase over the control rate value).

When 1 μM vasopressin was introduced to MDCK cells grown on a coverslip, no increase in metabolic rate was observed.

EXAMPLE 15

The addition of 10 mM MgCl to the control medium resulted in a 40-46%increase in metabolic rate with respect to the metabolic rate observedwhen the MDCK cells were exposed to medium only.

EXAMPLE 16

A silicon semiconductor electrode with a nylon mesh adhesively attachedto its upper surface, as described above, was prepared. CTLL cells wereflowed into the chamber at a flow rate of approximately 10 μL/min. Atthis flow rate some of the cells settled by gravity into the holes inthe nylon mesh. The flow rate was then increased to approximately 100μL/min and the cells remained in the holes. Cells could be visualized inthe microscope. The laser beam was directed to three different regionswhere cells were present and one region where no cells were present. Ateach site the change in pH with time was measured with the medium flowon and off. The slope of the pH change in μV/sec with the flow off minusthe same change with flow on was as follows:

    ______________________________________                                        (Flow Off-Flow On)                                                            Measurement μV/Sec    S.D.   Cells                                         ______________________________________                                        1           -9.7         .92    present                                       2           -6.2         .64    present                                       3           -4.4         .85    present                                       4           +1.7         .71    absent                                        ______________________________________                                    

EXAMPLE 17

This experiment was designed to demonstrate the possibility ofdetermining partial or complete identification of bacteria on the basisof their ability to change the pH of their immediate environment in thepresence of several potential carbon source compounds supplied one at atime in a serial manner to a single "catch" of bacteria. While thismethod of serial delivery of selective substrates has certain potentialadvantages, parallel delivery of selective substrates to separatecatches of bacteria could also be useful under certain circumstances.Furthermore, if bacteria are used for screening for antibioticsusceptibility or antibiotic activity, parallel delivery to separatecatches may well be more appropriate because of potential problems ofinteractions of drugs.

The bacteria, E. coli and Proteus mirabilis, were prepared assuspensions of about 5×10⁸ cells/ml in a growth medium and then dilutedinto a sterile test medium containing glucose (Bactopeptone, 2 g/L;NaCl, 5 g/L; K₂ HPO₄, 1.7 mM; glucose, 1 g/L; pH 7.3). With thecontrolling electrode piston in the raised position, as disclosed inpending U.S. patent application, Ser. No. 876,925, filed Jun. 20, 1986,one mL of bacterial suspension containing a known number of bacteria wasflowed through the device such that the cells were retained adjacent tothe silicon sensor surface by a Nucleopore membrane situated parallel tothe surface. The cells were pulsed with the same glucose containing testmedium, the piston was then lowered to decrease the volume surroundingthe cells, and the change in pH due to bacterial metabolism wasmonitored. The piston was then raised and a second test mediumcontaining urea in place of glucose was flowed through. After loweringthe piston the pH change was again monitored. This procedure wasrepeated with medium containing pyruvate as a carbon source.

FIG. 14 shows that E. coli respond by decreasing the pH in the presenceof all three media (each medium was tested three times in successionbefore testing the next medium). FIG. 15 shows that P. mirabilisdecreased the pH in the presence of glucose and pyruvate, but increasesthe pH in the presence of urea. FIG. 16 shows that the rate of pH changein glucose medium is dependent on the number of bacteria trapped in thedevice.

FIG. 18 shows E. Coli in a system that differs from that used for theexperiments shown in FIGS. 14 and 15 in that the cells were retained bya membrane, the plane of which was parallel to the flow of the mediumrather than perpendicular.

In FIG. 18, the E. Coli demonstrates an increase in pH change amplihidewith time consistent with an increase in the number of bacteria withtime. In addition to this apparent demonstration of bacterialreplication in the flow cell, FIG. 18 shows that E. Coli do not respondto an antibiotic that has low activity against gram negative bacteria(penicillin added at point A) but do respond to an antibiotic activeagainst gram negative bacteria (polymyxin added at point B).

EXAMPLE 18

CTLL cells were trapped in the flow chamber in rectangular wells.Referring to FIG. 17, an array 1 of 1000,μm long, 50 μm wide slots, with50 μm separation between slots, was cut into a 50 μm thick piece ofstainless steel foil 2 with an eximer laser. This was mounted in the 10μm deep flow chamber under pieces of 50 μm thick plastic 3 arranged topress the foil against the silicon semiconductor electrode surface whenthe cover slip was mounted. This left a channel about 1 mm wide and 5 μmdeep above the slots, thereby forming wells.

CTLL cells were settled into the wells at a linear flow velocity of themedium in the channel of no more than about 15 μm/sec. Alternatively,the wells could be loaded by flowing the medium faster than 15 μm/sec.until the cells were suspended above the wells, and then stopping theflow entirely for approximately 10 seconds while the cells sedimentedinto the wells. This flow/stop procedure was repeated until sufficientcells were trapped.

The cells remained in the wells for flow rates below approximately 100μm/sec, above which many were flushed out. Alternatively, momentaryinversion of the chamber caused the cells to sediment into the flowstream and be flushed away at lower flow rates.

The thin film of liquid between the foil and the silicon electrode, pluspossible corrosion of the stainless steel, makes the baseline potentialsomewhat unstable. This problem could be corrected by micromachining thewells directly into the silicon semiconductor electrode.

Metabolic rates of -30 to -60 μV/sec were obtained from CTLL cells. Thisis close to the range observed with adherent cells. Experiments withIL-2 similar to those in Example 11 suggested that after several hourswithout IL-2 the cells' metabolic rate had decreased to about 20-30% ofits control value; after several hours with IL-2 (20 U/ml) other cells'rate was over 50% of the control value. This is an indication that thepresence of IL-2 can be detected on the time scale of a few hours.Optimization of the apparatus may shorten that time considerably.

The present invention has been described in terms of certain preferredembodiments. Other embodiments not specifically described herein maynevertheless fall within the spirit or scope of the present invention orthe following claims.

We hereby claim as our invention:
 1. A method for detecting an effect ofa cell affecting agent on living cells comprising:(a) providing livingcells retained in a micro flow chamber having a means for continuous orintermittent flow of solutions or suspensions containing the cellaffecting agent in contact with the cells such that the amount of thecell affecting agent in contact with the cells can be controlled; (b)flowing a solution or suspension containing the cell affecting agentsuch that it comes into contact with the living cells thereby producinga change in pH of about 0.1 to 0.5 pH units in about 1 to 4 minutes and(c) repetitively stopping of the flow and repetitively measuring thechange in pH when the flow is stopped by a silicon semiconductorelectrode within about 100 μm of the living cells.
 2. The methodaccording to claim 1 wherein the living cells are adhered to the surfaceof the silicon semiconductor electrode.
 3. The method according to claim1 wherein the living cells are retained in a compartment in the vicinityof the silicon semiconductor by means of a membrane permeable to thecell affecting agent.
 4. The method according to claim 1 wherein theliving cells are adhered to a surface of the micro flow chamber.
 5. Themethod of claim 1 wherein the living cells are retained in wells in themicro flow chamber.
 6. The method of claim 4 wherein the cells areadherent.
 7. The method of claim 4 wherein the cells are adhered bymeans of agarose.
 8. The method of claim 1 wherein the cells areretained in a compartment in the micro flow chamber by means of amembrane permeable to the cell affecting agent.
 9. The method of claim 1wherein the solution or suspension is degassed prior to coming intocontact with the cells.
 10. The method of claim 1 wherein the effect ofthe cell affecting agent is measured at a plurality of sites on thesilicon semiconductor electrode.
 11. The method of claim 2 wherein thecells adhere to the surface of the silicon semiconductor electrode. 12.The method of claim 2 wherein the living cells are adhered to thesurface of the silicon semiconductor electrode by means of agarose. 13.The method of claim 1 wherein the cell affecting agent is a drug,hormone, toxin or immunological agent.
 14. A method for microbialidentification comprising:(a) providing a micro flow chamber adapted forcontinuous or intermittent flow of solutions or suspensions through themicro flow chamber wherein a portion of the micro flow chamber is asilicon semiconducting electrode and wherein the micro flow chamber hasa means for trapping microbes to be identified on or in the immediatevicinity of the silicon semiconducting electrode; (b) sequentiallyflowing a predetermined set of solutions or suspensions through themicro flow chamber where each solution or suspension contains one ormore ingredients that effect the rate of pH change of the microbe beingtested when contacted with the microbe to be identified; (c)repetitively stopping the flow and repetitively measuring the change inpH when the flow is stopped by means of the silicon semiconductingelectrode within about 100 μm of the cells and where the pH change isabout 0.1 to 0.5 pH units in about 1 to 4 minutes, (d) comparing themeasuring microbe response in pH change to the predetermined set ofsolutions or suspensions to the response of known bacteria therebyidentifying the bacteria to be identified.
 15. A method for screeningthe presence or activity of a drug comprising:(a) providing a micro flowchamber with a portion of the micro flow chamber being a siliconsemiconductor electrode, said micro flow chamber being adapted forcontinuous or intermittent flow of solutions or suspensions containing adrug to be tested through the micro flow chamber; (b) further providingliving cells responsive to the drug in the micro flow chamber in contactwith or in the immediate vicinity of the silicon semiconductorelectrode; (c) contacting the living cells with the drug to be tested byflowing a solution or suspension of the drug to be tested through themicro flow chamber and repetitively stopping the flow; and (d)repetitively measuring the change in pH when the flow is stopped bymeans of the silicon semiconductor electrode within about 100 μm fromthe cells and where the pH changes is about 0.1 to 0.5 pH units in about1 to 4 minutes.
 16. A method according to claim 15 wherein the drug isan antibiotic.
 17. A method for detecting a toxic substancecomprising:(a) providing a micro flow chamber wherein a portion of themicro flow chamber is a silicon semiconductor electrode, the micro flowchamber being adapted for continuous or intermittent flow of a solutionor suspension suspected of containing a toxic substance; (b) furtherproviding living cells responsive to the toxic substance being tested inthe micro flow chamber in contact with or in the immediate vicinity ofthe silicon semiconductor electrode; (c) contacting the living cellswith the solution or suspension suspected of containing the toxicsubstance by flowing the solution or suspension through the micro flowchamber and repetitively stopping the flow; and (d) repetitivelymeasuring the change in pH when the flow is stopped by means of thesilicon semiconductor electrode within about 100 μm of the cells andwherein the pH change is about 0.1 to 0.5 pH units in about 1 to 4minutes.